Substituted 4-dedimethylamino-4-hydroxy-4a, 12a-anhydrotetracyclines



United States Patent O 3,345,408 SUBSTITUTED 4-DED1METHY LAMINO-4-HY DROXY-4a,IZa-ANHYDROTETRACYCLINES Joseph John Hlavka, Tuxedo, and Panayota Bitha, New

York, N.Y., assignors to American Cyanamid Company, Stamford, Conn., a corporation of Maine N Drawing. Filed Nov. 30, 1964, Ser. No. 414,888

10 Claims. (Cl. 260-559) ABSTRACT OF THE DISCLOSURE This disclosure describes compounds of the class of substituted 4-dedimethylamino 4 hydroXy-4a,12a-anhydrotetracyclines useful as intermediates in the synthesis of physiologically active antibiotics of the tetracycline series.

OH I I \H/ CYONH2 0H 0 ('5 611 wherein R is hydrogen, chlorine or bromine and R is hydrogen or methyl.

The novel compounds of the present invention are obtainable as crystalline. materials having characteristic absorption spectra and high melting points. They are generally insoluble in Water but soluble in most organic solvents.

The novel compounds of the present invention may be readily prepared by dissolving an appropriate tetracycline methyl betaine [J.A.C.S., 80, 1654-1657 (1958)] in acetonitrile and refluxing the resulting solution under an atmosphere of nitrogen for from 1 to 2 hours. After the reaction is complete, the solvent is removed by evaporation to dryness and the product is isolated.

The novel compounds of the present invention are useful as intermediates in the synthesis of physiologically active antibiotics of the tetracycline series. Initially, a 4-dedimethylamino-4-hydroxy-4a,12a-anhydrotetracycline is converted to the corresponding 1,3,4,10,11,l2-hexahydroxynaphthacene-Z-carboxamide either by refluxing in a 30% solution of hydrobromic acid in acetic acid or by dissolving in concentrated sulfuric acid at room temperature. Either procedure results in the conversion of a 4-dedimethylamino-4-hydroXy-4a,IZa-anhydrotetra-- cycline to the corresponding 1,3,4, 10,11,12-hexahydroxynaphthacene-2-carboxamide. The 1,3,4,l0,11,12-hexahydroxynaphthacene-Z-carboxamide may then be biologically transformed to the tetracyclines as set forth in the following reaction scheme:

' ATCC 3,345,408 Patented Oct. 3, 1967 lit. R OH N(CH3)2 wherein R and R are as previously defined. This transformation is accomplished by adding a l,3,4,10,1l,12- heXahydroXynaphthacene-Z-carboxamide to a fermentation medium inoculated with a strain of a species of the genus Streptomyces which species is capable of producing one of the tetracyclines. Where R is hydrogen, and a non-halogenating strain of Streptomyces is employed, then R is also hydrogen in the product. Where R is hydrogen, and a 7-halogenating strain of the genus Streptomyces is employed, then R in the product is chlorine or bromine depending upon the conditions of the fermentation. Among the strains of S. aureofaciens which will introduce chlorine or bromine at the 7- position of the molecule, as well as accomplishing the 5a,6- and 4a,12a-dihydration and the introduction of the dimethylamino group at the 4-position, are the following. S. aureojacines ATCC 12554 ATCC 13189 ATCC 13899 ATCC 13900 NRRL B-1286 NRRL B-1287 NRRL B-1288 NRRL B-2209 NRRL B-2406 ATCC 12552 NRRL B-2407 ATCC 12553 NRRL 3013 A representative strain of the genus Streptomyces which is a non-halogenating strain, that is, which will not introduce halogen in the 7-position of the molecule, but which will accomplish the 5a,6- and 4a,12a-dihydration and the introduction of the dimethylamino group at the 4-position, is S. aureofaciens NRRL 3014.

The conditions of the fermentation for the biological conversion of a 1,3,4,10,11,12-hexahydroxynaphthacene- 2-carboxamide to a tetracycline are generally the same as set forth in US. Patent 2,482,055 to Duggar, US. Patent 2,734,018 to Minieri et al. and US. Patent 2,878,289 to McCormick et al. and which, in turn, are generally the same as for the presently known methods for producing various tetracyclines by fermentation. That is, the fermentation medium contains the usual nutrients and mineral substances. Suitable nutrients include any assimilable source of carbon, such as the polysaccharides or starches, or polyalchols such as glycerol may be used. An assimilable source of nitrogen may be supplied through the use of porteins, protein hydrolysates, urea, corn steep liquor, meat extracts, peptone, distillers solubles, fish meal and other conventional substances. The common anions and cations are supplied in the form of their non-toxic salts. Trace elements such as manganese, cobalt, zinc, copper, etc., are obtained either as impurities in the above compounds, or through the use of tap water or by specifically adding solutions especially enriched with these trace elements.

The other general conditions of the fermentation such as hydrogen ion concentration, temperature, time rate of aeration, preparation of the inoculum, sterilization, inoculation and the like are conventional and are similar to those for the production of other tetracyclines as set ATCC ATCC ATCC ATCC ATCC ATCC ATCC 10762a 107621) 10762g 10762i 11989 12416b 124160 12416d ATCC 12551 J forth in the aforementioned US. patents to Duggar, Minieri et al. and McCormick et a1.

When a 7-halogenating strain of the genus Streptomyces is employed with .a 1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide wherein R is hydrogen, it is necessary only to modify the fermentation medium so that it contains at least 10 parts per million and preferably 1000- 1500 parts per million of chloride ions when the 7-chloro substituent is desired, or a like amount of bromide ions when the 7-bromo substituent is desired.

After the fermentation has been continued for a suitable time, for example, from 12 to 96 hours, and the transformation of the 1,3,4,10,11,12-hexahydroxynaphthacene-2-carboxamide to the desired tetracycline is substantially complete, the tetracycline product may be isolated from the fermentation mash in any convenient manner. The isolation process may be selected from any of the numerous isolation techniques now well known in the art.

The 1,3 ,4,10,1 1,12-hexahydroxynaphthacene-Z-carboxamide starting material may be added at any desired concentration, although for practical reasons a 1,3,4,10,11, 1Z-hexahydroxynaphthacene-2-carboxamide substrate at a concentration of up to about 10 grams per liter of medium is satisfactory although higher concentrations may be used with some sacrifice in yield. The addition of the 1,3,4 10,1l,12-hexahydroxynaphthacene-2-carboxamide starting material may be accomplished in any suitable manner so long as it promotes contact of the 1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide with the biological medium. To this end, it is preferred to add the 1,3,4,10,11, 12-hexahydroxynaphthacene-2-carboxamide starting material in a solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetramethylenesulfoxide and N-methylpyrrolidone. However, dimethylsulfoxide is preferred and a solution of magnesium acetate in dimethylsulfoxide is the most preferred solvent for the 1,3,4,10, 11, 12 hexahydroxynaphthacene-2-carboxamide starting material. Solutions of the 1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamides must be protected from air as the compounds are readily oxidized in solution.

Since the novel compounds of the present invention may be converted to the corresponding l,3,4,10,11,12- hexahydroxynaphthacene-Z-carboxamides which are more stable to electrophilic substitution under acid conditions, the substituents at the 7- and/or 9-positions thereof may be varied (i.e. Cl, Br, N and the resulting compound may then be bioconverted to the corresponding substituted tetracycline. In addition, the l,3,4,10,11,12-hexahydroxynaphthacene-2-carboxamides may be reduced with phenol and hydriodic acid to the corresponding 1,3,10,11,12-pentahydroxynaphthacene-Z-carboxamides which could be first chemically modified and then bioconverted to the corresponding tetracycline.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Preparation of 6-demethyl-7-chl0r0tetracycline methiodide The 6-dernethyl-7-chlorotetracycline free base, 3 g., is dissolved in 60 ml. of hot tetrahydrofuran and the solution is filtered. To the filtrate is added 9 ml. of methyl iodide and the mixture is allowed to stand at room temperature for 6 days. A small amount of amorphous precipitate is filtered, washed with ether and dried to afford 396 mg. of product. The filtrate is diluted with about 200 m1. of ether and the light yellow solid is isolated by filtration. Yield: 3.18 g. This material is crystallized from methanol as follows: The solution of 1 g. of product in 4 ml. of methanol is stirred at room temperature for 10 minutes. Crystalline precipitate is filtered and dried in pistol at 62 C. for two hours. This product has the following properties:

0.1 N HCl:

k =228 ma [log 6 4.41], 270 m [log a 4.14],

373 ma [10g 6 4.03]. 0.1 M borate:

k =225 ma [log 6 4.41], 290 mu [log e 4.09],

375 mp. [log e 4.20].

EXAMPLE 2 Preparation of 6-demethyl-7-chl0r0tetracycline methyl betaine The 6-demethyl-7-chlorotetracycline methiodide, 200 mg., is dissolved in 5 ml. of water. The solution is adjusted immediately to pH 6.8 with a few drops of 20% sodium acetate solution. One ml. of methanol is added to get the precipitated solid into solution which is then stirred for 10 minutes at room temperature. Methanol is stripped off under vacuum and the crystalline product which precipitates from water is filtered and dried at 62 C. overnight to afford 143 mg. of product.

EXAMPLE 3 Preparation of 4-dedimethylamin0-4-hydr0vcy-4ag12aanhydro-6-demethyl-7-chl0r0tetracycline The 6-demethyl-7-chlorotetracycline betaine, 3 g., is suspended in 500 ml. of acetonitrile and nitrogen is bubbled through for 15 minutes. The suspension is then heated under nitrogen atmosphere to the boiling point of acetonitrile and refluxed for 4 hours. A small amount of solid does not go in solution. Heat is removed and bubbling of nitrogen is continued until most of the trimethylamine vapors are stripped off (about 10 minutes). The undissolved material is filtered. The filtrate is evaporated to dryness to afford 2.1 g. of a mixture which is purified as follows: 1.5 g. of this material is dissolved in 500 ml. of methanol. A yellow product crystallizes out within a few minutes. It is allowed to stand at room temperature for one hour and then filtered. It is washed well with ether and dried in pistol at 62 C. Yield: 381 mg.

0.1 N HCl:

A =285 m [log 6 3.86], 400 m [log 6 4.05]. 0.1 M borate:

k =255 ma [log e 4.20], 458 mu [log e 4.29].

EXAMPLE 4 Preparation of 7-chl0ro-13,4,10,11,12-hexahydroxynaphthacene-Z-carb0xamide conc. H SO 1% borate:

A =285 ma [log 6 4.22], 318 mn [log e 4.61],

481 m [log e 4.16], 534 my. [log 6 3.96].

EXAMPLE 5 Preparation of 4-dedimethylamin0-4-hydr0xy-4a, IZa-anhydrotetracycline A 2.0 gram portion of tetracycline methyl betaine is suspended in 500 ml. of acetonitrile. Nitrogen is bubbled through this suspension for 15 minutes. The suspension is then heated under nitrogen atmosphere to the boiling point of acetonitrile and refluxed for 1 /2 hours. The heat is then removed and the bubbling of nitrogen is continued for an additional 15 minutes until all of the trimethylamine vapors have been stripped off. An orange crystalline product precipitates upon cooling. This precipitate is collected by filtration, washed with acetonitrile and dried in a vacuum desiccator toaiford mg. of product.

0.1 N HCl A =370 ma [log e 3.76], 485 m [log 6 4.17]

EXAMPLE 6 Conversion of 4-dedimethylamin0-4-hydroxy-4w,12a-anhydrotetracycline to 6-methyl-1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide Biological conversion of I,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide t0 7-chloro-6demethyltetracycline Spores of S. aurofaciens NRRL 3013 are washed from an agar slant with sterile distilled water to form a suspension containing 60'80 spores per ml. A 0.33 ml. portion of this suspension is used to inoculate an 8 inch test tube containing 8 ml. of a medium prepared according to the following formulation:

Grams Sucrose 30 Ammonium sulfate 2 Calcium carbonate 7 Cornsteep liquor 20 Tap water, q.s. to 1000 milliliters.

Prior to inoculation, the medium is sterilized by autoclaving for 20 minutes under a pressure of pounds per square inch. The inoculated tube is then incubated for 24 hours at 28 C. on a reciprocating shaker operating at 116 oscillations per minute, whereby an inoculum of the S. aureofaciens is obtained. A fermentation medium of the following composition is prepared:

grams 6.7

(NI-I SO CaCO d0 9.0 Co Cl -6H O milligrams 5.0 NH CI grams 2.0 MnSO (70% technical grade) do 0.10 Cornsteep liquor do 25.0 Starch do 52.5 Corn flour do 14.5

Tap water, q.s. to 1000 milliliters.

After sterilization of this medium in an autoclave for minutes at a pressure of 15 pounds per square inch, a ml. portion in a 250 ml. Erlenmeyer flask is inoculated with a 1.0 ml. portion of the S. aureofaciens inoculum. The fermentation is carried out at 25 C. for 24 hours on a rotary shaker operating at 180' revolutions per minute. At this time a partially fermented mash of S. aureofaciens NRRL 3013 is transferred to an individual flask containing a solution of 5.45 mg. of 1,3,4,10,11,12- hexahydroxynaphthacene-Z-carboxamide in mg. of magnesium acetate and 1 ml. of dimethylsulfoxide. The fermentation is then continued on the rotary shaker for an additional 72 hours at 28 C. At this time, analysis of the mash shows the presence of 17 micrograms per ml. of 7-chloro-6-demethyltetracycline. A control flask run in exactly the same manner but omitting the 1,3,4,10,11, 12 hexahydroxynaphthacene-Z-caboxamide shows no 7- chloro-6-demethyltetracycline.

EXAMPLE 8 Preparation of 4-dedimethylamino-4-hydr0xy-4a,12a-anhydro-7-chlorotetracycline The 7-chlorotetracycline methyl betaine, 3.44 g., is suspended in 630 ml. of. acetonitrile and nitrogen is bubbled through for 15 minutes. The suspension is then heated under nitrogen atmosphere to the boiling point of acetonitrile and the resulting solution is refluxed for one and a half hours. Heat is removed and bubbling of nitrogen is continued until most of trimethylamine vapors are stripped olf (about 10 minutes). A small amount of solid precipitates out upon cooling which is filtered off. The filtrate is evaporated to dryness. Solid is received in 500 ml. of ether and refluxed for 30- minutes. Undissolved solid is filtered (1.713 g.). Filtrate which contains the desired products is evaporated to dryness (wt.=l.192 g.) and purified by partition column chromatography using the system: heptane-ethyl acetate-methanol-water (-20-174). The product is obtained in two tautomeric forms which gives the same ultraviolet spectra in 0.1 N HCl as follows:

Xmax' 250 mn- [log. 6 4.32], 362 my [log 6 3.90].

EXAMPLE 9 Preparation of 7-chloro-6-methyl-1,3,4,10,11,IZ-hexahydroxynaphthacene-Z-carboxamide The 4-dedimethylamino-4-hydroxy-4a,12a anhydro-7- chlorotetracycline, 20 mg, is dissolved in 4 ml. of a solution of 32% hydrobromic acid in acetic acid and the red solution is evaporated immediately to dryness under vacuum. The solid is received in ether, filtered and dried in vacuo to afford 16.7 mg. of product analyzing as follows:

A =27O [log 6 4.32], 310 [10g 6 4.39], 508 [10g The spectrum is taken in concentrated H 80 containing 1% sodium borate.

EXAMPLE 10 Biological conversion of 6-methyl-1,3,4,10,11,12-hexahydroxynaphthacene-Z-carboxamide to 7-chl0rotetracycline Spores of S. auerofaciens NRRL 3013 were washed from an agar slant with sterile distilled water to form a suspension containing 60-80 10 spores per ml. A 0.33 ml. portion of this suspension was used to inoculate an 8 inch test tube containing 8 ml. of an inoculum medium prepared according to the following formulation:

. Grams Sucrose 30 Ammonium sulfate 2 Calcium carbonate 7 Cornsteep liquor 20 Tap water, q.s. to 1000 milliliters.

After sterilization of this medium, 25 ml. portions in 250 ml. Erlenmeyer flasks were inoculated with 1.0 ml. portions of the S. aureofaciens NRRL 3013 inoculum. The fermentation was carried out at 28 C. for 24 hours on a rotary shaker operating at revolutions per minute. At this time each mash portion was transferred to an individual flask containing a solution of 10.7 mg. of 6-1nethyl- 1,3,4, 10,1 1,12-hexahydroXynaphthacene-2-carboxamide in a mixture of 30 mg. of magnesium acetate and 1 ml. of dimethylsulfoxide. The fermentation was continued on the rotary shaker for an additional 96 hours at 28 C. At

this time, biological assays of the mash indicated the presence of antibacterial activity corresponding to 264 micrograms of 7-chlorotetracycline per ml. This corresponds to a yield of 49% based on the compound added. The identity of the product as 7-chlorotetracycline was confirmed by paper chromatography in a butanol-pH3 phosphate buffer system. A control flask run in exactly the same manner but with the addition of only 30 mg. of magnesium acetate and 1 ml. of dimethylsulfoxide and no 6- methyl 1,3,4,10,11,12 hexahydroxynaphthacene-Z-carboxamide, showed no 7-chlorotetracycline.

EXAMPLE ll Biological conversion of 7-chl0r0-6-methyl-1,3,4,10,11,12- hexahydroxynaphthacene-Z-carboxam ide to 7-ch lorotetracycline utilizing a non chlorinating strain of S. aureofaciens Spores of non-chlorinating S. aureofaciens NRRL 3014 were washed from an agar slant with sterile distilled water to form a suspension containing 60-80 10 spores per ml. A 0.33 ml. portion of this suspension was used to inoculate an 8 inch test tube containing 8 ml. of a medium prepared according to the following formulation:

Grams Sucrose 30 Ammonium sulfate 2 Calcium carbonate 7 Cornsteep liquor 20 Tap water, q.s. to 1000 milliliters.

Prior to inoculation, the medium was sterilized by autoclaving for 20 minutes under a pressure of 15 pounds per square inch. The inoculated tube was then incubated for 24 hours at 28 C. on a reciprocating shaker operating at 116 oscillations per minute, whereby an inoculum of the S. aureofaciens was obtained. A fermentation medium of the following composition was prepared.

Tap water, q.s. to 1000 milliliters.

After sterilization of this medium in an autoclave for 20 minutes at a pressure of 15 pounds per square inch, 25 ml. portions in 250 ml. Erlenmeyer flasks were inoculated with 1.0 ml. portions of the S. aureofaciens inoculum. The fermentation was carried out at 25 C. for 24 hours on a rotary shaker operating at 180 revolutions per minute. At this time each mash portion Was transferred to an individual flask containing a solution of 10 mg. of 7- chloro 6 methyl-1,3,4,l0,11,12-hexahydroxynaphthacene-Z-carboxamide in a mixture of 10 mg. of magnesium acetate and 1 ml. of dimethylsulfoxide. The fermentation was then continued on the rotary shaker for an additional 96 hours at 28 C. At this time analysis of the mash showed the presence of 10 micrograms per ml. of 7-chlorotetracycline. A control flask run in exactly the same manner but omitting the 7-chloro-6-methyl-1,3,4, 10,1 1, l 2-hexahydroxynaphthacene-Z-carboxamide showed no 7-chlorotetracycline.

EXAMPLE 12 Biological conversion of 7-chlor0-1,3,4,10,11,12-hexahydroxynaphthacene 2 carboxamide to 7-chlor0-6-demethyltetracycline utilizing a non-chlorinating strain of S. aureofaciens The procedure of Example 11 was followed with these exceptions: The partially fermented (24 hours) mash was transferred to flasks containing a solution of 10 mg, of

Biological conversion of 7-chlor0-6-methyl-1,3,4,10,11, 12-hexahydroxynaphthacene-Z-carb0xamide to 7-chlorotetracycline utilizing a chlorinating strain of S. aureofaciens The procedure of Example 11 was followed with these exceptions: Spores of chlorinating S. aureofaciens NRRL 3013 were washed from an agar slant with sterile distilled water to form a suspension containing 6080 10 spores per ml. A 0.33 ml. portion of this suspension was used to inoculate an 8 inch test tube containing 8 ml. of the medium described in Example 11. The partially fermented (24 hours) mash (prepared as in Example 11) was transferred to flasks containing a solution of 10 mg. of 7-chloro-6-methyl-1,3 ,4,10,1 1,12-hexahydroxynaphthacene-2-carboxamide in a mixture of 10 mg. of magnesium acetate and 1 ml. of dimethylsulfoxide. The fermentation was then continued on the rotary shaker for an additional 96 hours at 28 C. At this time, analysis of the mash showed the presence of 10 micrograms per ml. of 7-chlorotetracycline. A control flask run in exactly the same manner but omitting the 7-chloro-6-methyl-1,3,4, 10,11,12-hexahydroxynaphthacene-Z-carboxamide showed no 7-chlorotetracycline.

EXAMPLE l4 Biological conversion of 7-chloro-1,3,4,10,11,12-hexahydroxynaphthacene 2 carboxamide to 7-chloro-6-demethyltetracycline utilizing a chlorinating strain of S. aureofaciens The procedure of Example 13 was followed with these exceptions: The partially fermented (24 hours) mash was transferred to flasks containing a solution of 10 mg. of 7 chloro-1,3,4,10,l1,12-hexahydroxynaphthacene-Z-carboxamide in a mixture of 10 mg. of magnesium acetate and 1 ml. of dimethylsulfoxide. The fermentation was then continued on the rotary shaker for an additional 96 hours at 28 C. At this time analysis of the mash showed the presence of 9 micrograms per ml. of 7-chloro-6-demethyltetracycline. A control flask run in exactly the same manner but omitting the 7-chloro-l,3,4,10,11,12- hexahydroxynaphthacene-2-carboxamide showed no 7- chloro-6demethyltetracycline.

What is claimed is:

1. A compound of the formula:

wherein R is selected from the group consisting of hydrogen, chlorine and bromine, and R is selected from the group consisting of hydrogen and methyl.

2. 4 dedimethylamino 4-hydroxy-4a,l2a-anhydrotetracycline.

3. 7 chloro 4 dedimethylamino-4-hydroxy-4a,12aanhydrotetracycline.

4. 7 bromo 4dedimethylamino-4-hydroxy-4a,12aanhydrotetracycline.

5. 6 demethyl 4 dedimethylamino-4-hydroxy-4a, l2a-anhydrotetracycline.

r 'V r l-OH CONH2 I H H 0H 0 0 OH wherein R is selected from the group consisting of hydrogen, chlorine and bromine and R is selected from the group consisting of hydrogen and methyl; which comprises refluxing a compound of the formula:

wherein R and R are as hereinabove defined, in acetonitrile under an inert atmosphere.

9. The process of preparing compounds of the formula:

CONH:

wherein R is selected from the group consisting of hydrogen, chlorine and bromine and R is selected from the group consisting of hydrogen and methyl; which comprises refluxing a compound of the formula:

' R R OH OH CONH2 wherein R and R are as hereinabove defined, in a strong mineral acid.

10. The process of preparing compounds of the formula:

R R OH \/CONHa l OH )11 OH 6H wherein R is selected from the group consisting of hydrogen, chlorine and bromine and R is selected from the group consisting of hydrogen and methyl; which comprises dissolving a compound of the formula:

OH O OH wherein R and R are as hereinabove defined, in a strong mineral acid.

References Cited UNITED STATES PATENTS 3,201,424 8/1965 McCormick et a1. 260-659 NICHOLAS S RIZZO, Primary Examiner 

1. A COMPOUND OF THE FORMULA: 